Methods for cloning recombinant DNA began with restriction enzymes and ligase to join vector replicon such as a plasmid with target passenger DNA to be cloned. PCR allows the ready generation of the target and/or vector DNA fragments with short overlapping sequence homology at their ends, enabling so-called ligase independent cloning (LIC). These methods obviate to lesser or greater extents that any particular sequence or recognition site be present at the ends of the DNA regions to be joined. An early LIC method required a certain sequence simplicity at the ends to be joined (only 3 bases complexity(1)) and single-strand ends were produced by the plus method (2) which involves treatment with 1 dNTP and the 3′-exonuclease of a DNA polymerase.
Other LIC methods make use of specially prepared, special vectors with special site-specific sites (3-5) or 2 sizes of both vector and target, followed by complete denaturation and reannealing (6).
Ribocloning (7) requires special PCR primers for both target and vector that end in a ribo-U or a ribo-C at their 3′-ends, treatment by RNAse in low salt, and removal of the released primers for maximum efficiency.
Some LIC methods use an overlap of normal DNA nucleotides, but employ digestion on the 3′-end by any of several exonucleases, such as ExoIII (8), T4 DNA polymerase (9-11), or vaccinia virus DNA polymerase (12), creating 5′-sticky end single strands.
It was reported in one of the T4 DNA polymerase methods (10) that the 5′-exonucleases encoded by phage T7 and lambda are less effective and reproducible.